ABSTRACT
OBJECTIVES: To characterise the genotypes of multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) isolated in Algeria, where there is a low MDR-MTB incidence rate. METHODS: Ten MDR isolates and one resistant to isoniazid were investigated by PCR-Sanger sequencing for 10 loci involved in resistance. Amplicon-based next generation sequencing (NGS) of 15 loci was additionally performed on isolates harbouring novel mutations. RESULTS: Sanger and amplicon-NGS provided the same results as with GenoType kits. Mutations known to be associated with resistance were described for most isolates: rpoB S531L in seven of 10 rifampicin-R MTB isolates, katG S315T in nine of 11 isoniazid-R, and promoter inhA c-15t in three of 11, embB M306V or M306I in two of two ethambutol-R, rpsL K43R in four of eight or rrs a514c associated with gidB L16R in streptomycin-R, gyrA A90V in the ofloxacin-R pre-XDR isolate. New and rare mutations were also described in rpoB (deletion 512-513-514), katG (S315R, M126I/ R496L), gidB (V124G, E92A, V139A, G37V), and gyrA (P8A). Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) profiles were similar for three isolates (lineage Cameroon), indicating a possible clonal diffusion in epidemiologically unrelated patients. CONCLUSIONS: Resistant MTB isolates in Algeria harbour resistance genotypes similar to other countries, but some rare patterns may result from selection and transmission processes inherent to the country.
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/microbiology , Adult , Aged , Aged, 80 and over , Algeria , Genotype , High-Throughput Nucleotide Sequencing , Humans , Microbial Sensitivity Tests , Middle Aged , Mutation , Tuberculosis, Pulmonary/microbiology , Young AdultABSTRACT
Mycobacterium abscessus is an emerging pathogen against which clarithromycin is the main drug used. Clinical failures are commonly observed and were first attributed to acquired mutations in rrl encoding 23S rRNA but were then attributed to the intrinsic production of the erm(41) 23S RNA methylase. Since strains of M. abscessus were recently distributed into subspecies and erm(41) sequevars, we investigated acquired clarithromycin resistance mechanisms in mutants selected in vitro from four representative strains. Mutants were sequenced for rrl, erm(41), whiB, rpIV, and rplD and studied for seven antibiotic MICs. For mutants obtained from strain M. abscessus subsp. abscessus erm(41) T28 sequevar and strain M. abscessus subsp. bolletii, which are both known to produce effective methylase, rrl was mutated in only 19% (4/21) and 32.5% (13/40) of mutants, respectively, at position 2058 (A2058C, A2058G) or position 2059 (A2059C, A2059G). No mutations were observed in any of the other genes studied, and resistance to other antibiotics (amikacin, cefoxitin, imipenem, tigecycline, linezolid, and ciprofloxacin) was mainly unchanged. For M. abscessus subsp. abscessus erm(41) C28 sequevar and M. abscessus subsp. massiliense, not producing effective methylase, 100% (26/26) and 97.5% (39/40) of mutants had rrl mutations at position 2058 (A2058C, A2058G, A2058T) or position 2059 (A2059C, A2059G). The remaining M. abscessus subsp. massiliense mutant showed an 18-bp repeat insertion in rpIV, encoding the L22 protein. Our results showed that acquisition of clarithromycin resistance is 100% mediated by structural 50S ribosomal subunit mutations for M. abscessus subsp. abscessus erm(41) C28 and M. abscessus subsp. massiliense, whereas it is less common for M. abscessus subsp. abscessus erm(41) T28 sequevar and M. abscessus subsp. bolletii, where other mechanisms may be responsible for failure.
